The quest to visualize ever smaller, fainter structures has driven much scientific progresses. Spatial resolution and contrast, essential factors in imaging, are limited by the wavelength and the intensity noise, respectively. While shorter wavelengths (X-rays, electron beams) can improve resolution and fluorescent labeling can increase contrast, these benefits come at the expense of harmful radiation and invasive sample preparation.
The “State of the Art” in conventional optical microscopy is limited by the wavelength. There have been attempts to “break the barrier” of the wavelength. These attempts reach fraction of wavelength resolution. The general trend for improved resolution has been to develop sources and techniques at much shorter wavelengths. The shorter the wavelength, the more harmful the radiation. Most of the techniques with shorter wavelength radiation require complex environmental condition (for instance, in vacuum for the electron microscope), and most generally sophisticated sample preparation.
The resolution of a traditional optical microscope is limited by the maximum spatial frequency that can be transmitted by a microscope objective, leading to a resolution limit of λ/(2nA), where nA is the numerical aperture of the microscope objective, which is ≈250 nm for visible light microscopy. Current methods that seek to build three-dimensional reconstructions of a sample using the diffraction of light by index of refraction variations, such as Optical Diffraction Tomography (ODT), are ultimately limited by this resolution limit. In addition, in living cells, many structures of interest are either too small or do not have an index difference large enough for suitable contrast. For this reason, fluorescence microscopy has become the most widely used optical technique for studying living cells.
In fluorescence microscopy, the cellular component of interest is labeled with a fluorophore for specificity and contrast. Upon excitation with (usually) visible light, the fluorophore is excited and emits at a longer wavelength, a Stoke's shifted emission that is collected by a microscope objective and separated from the excitation light. For studies of extra-cellular membrane components, fluorescent probes can be pre-conjugated to ligands or antibodies. Ligand and antibody probes have difficulty passing the cell membrane. Thus for studies of cytoplasmic or nuclear components, cells are typically transfected to express chimeric proteins-proteins containing an additional fluorescent protein such as Green Fluorescent Protein (GFP) or other variants. As expression levels in transfected cells can vary significantly, it becomes difficult to label more than two intra-cellular components simultaneously. In all cases, labeling procedures must be verified to not interfere with normal function.
During imaging, excited fluorophores can undergo a transition from an excited singlet state to a much longer lived triplet state. This long lived state has an increased probability to interact with molecular oxygen, which can both irreversibly chemically alter the fluorophore (photo-bleaching) and create a free radical singlet oxygen that can further damage other molecules in the cell. The destruction of the fluorophore by photo-bleaching limits the amount of emitted and collected photons from each probe, placing restrictions on long term studies and the super-resolution techniques discussed below, which require high signal to noise.
Several techniques have been demonstrated that improve the resolution of the fluorescence microscope. Techniques such as 4π microscopy and I5M provide a near uniform lateral and z-axis resolution of near 100 nm, using coherent collection by two opposing high-numerical aperture microscope objectives and deconvolution. However, these techniques still rely on a linear response of the probe to excitation light, and merely extend the resolution limit, but do not break it. Both Stimulated Emission Depletion (STED) and saturated patterned excitation microscopy make use of the non-linearity inherent in fluorescence saturation to break the diffraction limit, and both have demonstrated a resolution of better than 50 nm. Recently, several techniques that rely on the photo-activable or photo-switchable fluorescent probes have been demonstrated to give better than 20 nm resolution by building images through the localization of sparse sets of individual fluorescent probes. Localization of single probes can be performed with accuracy of better than 10 nm with relatively few collected photons (<1000), however repetitively preparing (by photo-activation) and imaging a sparse set of emitters leads to collection times of minutes at best. All of these techniques require a fluorescent probe, are limited by signal to noise and are ultimately constrained by photo-bleaching.